A systematic analysis of the structure, function, and regulation of the pyruvate and alpha-ketoglutarate dehydrogenase complexes from microorganisms and from mammalian tissues is continuing. Activity of the mammalian pyruvate dehydrogenase complex is regulated by phosphorylation and dephosphorylation catalyzed, respectively, by a MgATP2--requiring kinase and a Mg2 ion-requiring phosphatase. The site of this covalent regulation is the pyruvate dehydrogenase component of the complex. Pyruvate dehydrogenase exists as a tetramer, alpha2 beta2. Phosphorylation occurs on three serine residues in the alpha-subunit. The amino acid sequences around the three phosphorylation sites have been determined. Phosphorylation proceeds markedly faster at Site 1 than at Sites 2 and 3, and phosphorylation at Site 1 correlates closely with inactivation of pyruvate dehydrogenase. Pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase have been isolated in a homogenous state from bovine kidney and heart mitochondria. The subunit structure and regulatory properties of the kinase and the phosphatase are under investigation.